HEMOSTASIS: PLATELET FUNCTION, BLOOD COAGULATION, AND FIBRINOLYSIS

Hemostasis is the cessation of blood loss from a damaged vessel. Platelets first adhere to macromolecules in the subendothelial regions of the injured blood vessel; they then aggregate to form the primary hemostatic plug. Platelets stimulate local activation of plasma coagulation factors, leading to generation of a fibrin clot that reinforces the platelet aggregate. Later, as wound healing occurs, the platelet aggregate and fibrin clot are degraded.

Coagulation involves a series of zymogen activation reactions,. At each stage, a precursor protein, or zymogen, is converted to an active protease by cleavage of one or more peptide bonds in the precursor molecule. The components at each stage include a protease from the preceding stage, a zymogen, a nonenzymatic protein cofactor, Ca2+, and an organizing surface that is provided by a phospholipid emulsion in vitro or by platelets in vivo. The final protease generated is thrombin (factor IIa).

Conversion of Fibrinogen to Fibrin. Fibrinogen is a 330,000-dalton protein that consists of three pairs of polypeptide chains (designated Aa, Bb, and g) covalently linked by disulfide bonds. Thrombin converts fibrinogen to fibrin monomers by cleaving fibrinopeptides A (16 amino acid residues) and B (14 amino acid residues) from the amino-terminal ends of the Aa and Bb chains, respectively. Removal of the fibrinopeptides allows the fibrin monomers to form a gel, which is the end point of in vitro assays of coagulation (see below). Initially, the fibrin monomers are bound to each other noncovalently. Subsequently, factor XIIIa catalyzes an interchain transglutamination reaction that cross-links adjacent fibrin monomers to enhance the strength of the clot.

Structure of Coagulation Protease Zymogens. The protease zymogens involved in coagulation include factors II (prothrombin), VII, IX, X, XI, XII, and prekallikrein. About 200 amino acid residues at the carboxyl-terminal end of each zymogen are homologous to trypsin and contain the active site of the protease. In addition, 9 to 12 glutamate residues near the amino-terminal ends of factors II, VII, IX, and X are converted to g-carboxyglutamate (Gla) residues during biosynthesis in the liver. The Gla residues bind Ca2+ and are necessary for the coagulant activities of these proteins.

Nonenzymatic Protein Cofactors. Factors V and VIII are homologous 350,000-dalton proteins. Factor VIII circulates in plasma bound to von Willebrand factor, while factor V is present both freely in plasma and as a component of platelets. Thrombin cleaves V and VIII to yield activated factors (Va and VIIIa) that have at least 50 times the coagulant activity of the precursor forms. Factors Va and VIIIa have no intrinsic enzymatic activity but serve as cofactors that increase the proteolytic efficiency of Xa and IXa, respectively. Tissue factor (TF) is a nonenzymatic lipoprotein cofactor that greatly increases the proteolytic efficiency of VIIa. It is present on the surface of cells that do not normally contact plasma (e.g., macrophages and smooth muscle cells) and initiates coagulation outside a broken blood vessel. Monocytes and endothelial cells also may express tissue factor when exposed to a variety of stimuli, such as endotoxin, tumor necrosis factor, and interleukin-1. Thus these cells may be involved in thrombus formation under pathological circumstances. High-molecular-weight kininogen is a plasma protein that serves as the cofactor for XIIa when clotting is initiated in vitro in the activated partial thromboplastin time (aPTT) test.

Activation of Prothrombin. Factor Xa cleaves two peptide bonds in prothrombin to form thrombin. Activation of prothrombin by Xa is accelerated by Va, phospholipids, and Ca2+. When these components are all present, prothrombin is activated nearly 20,000 times faster than the rate achieved by Xa and Ca2+ alone. The maximal rate of activation occurs only when prothrombin and Xa both contain Gla residues, and therefore have the ability to bind to phospholipids. Purified platelets can substitute for phospholipids and Va to facilitate activation of prothrombin in vitro, provided that the platelets are stimulated to release endogenous platelet factor Va or that factor Va is added exogenously to unstimulated platelets. The surface of platelets that are aggregated at the site of hemostasis concentrates the factors required for prothrombin activation.

Initiation of Coagulation. Coagulation is initiated in vivo by the extrinsic pathway. Small amounts of factor VIIa in the plasma bind to subendothelial tissue factor following vascular injury. Tissue factor accelerates activation of factor X by VIIa, phospholipids, and Ca2+ about 30,000-fold. VIIa also can activate IX in the presence of tissue factor, providing a convergence between the extrinsic and intrinsic pathways.

Clotting by the intrinsic pathway is initiated in vitro when XII, prekallikrein, and high-molecular-weight kininogen interact with kaolin, glass, or another surface to generate small amounts of XIIa. Activation of XI to XIa and IX to IXa follows. IXa then activates X in a reaction that is accelerated by VIIIa, phospholipids, and Ca2+. Activation of factor X by IXa appears to occur by a mechanism similar to that for activation of prothrombin and may also be accelerated by platelets in vivo. Activation of factor XII is not required for hemostasis, since patients with deficiency of XII, prekallikrein, or high-molecular-weight kininogen do not bleed abnormally, even though their aPTT values are prolonged. Factor XI deficiency is associated with a variable and usually mild bleeding disorder. The mechanism for activation of factor XI in vivo is not known, although thrombin activates factor XI in vitro.

Fibrinolysis and Thrombolysis

The fibrinolytic system dissolves intravascular clots as a result of the action of plasmin, an enzyme that digests fibrin. Plasminogen, an inactive precursor, is converted to plasmin by cleavage of a single peptide bond. Plasmin is a relatively nonspecific protease; it digests fibrin clots and other plasma proteins, including several coagulation factors. Therapy with thrombolytic drugs tends to dissolve both pathological thrombi and fibrin deposits at sites of vascular injury. Therefore, the drugs are toxic, producing hemorrhage as a major side effect.

The fibrinolytic system is regulated such that unwanted fibrin thrombi are removed, while fibrin in wounds persists to maintain hemostasis (Lijnen and Collen, 2001). Tissue plasminogen activator (t-PA) is released from endothelial cells in response to various signals, including stasis produced by vascular occlusion. It is rapidly cleared from blood or inhibited by circulating inhibitors, plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2, and thus exerts little effect on circulating plasminogen. t-PA binds to fibrin and converts plasminogen, which also binds to fibrin, to plasmin. Plasminogen and plasmin bind to fibrin at binding sites located near their amino termini that are rich in lysine residues (see below). These sites also are required for binding of plasmin to the inhibitor a2-antiplasmin. Therefore, fibrin-bound plasmin is protected from inhibition. Any plasmin that escapes this local milieu is rapidly inhibited. Some a2-antiplasmin is bound covalently to fibrin and thereby protects fibrin from premature lysis. When plasminogen activators are administered for thrombolytic therapy, massive fibrinolysis is initiated, and the inhibitory controls are overwhelmed.

Coagulation in Vitro. Blood clots in 4 to 8 minutes when placed in a glass tube. Clotting is prevented if a chelating agent such as ethylenediaminetetraacetic acid (EDTA) or citrate is added to bind Ca2+. Recalcified plasma clots in 2 to 4 minutes. The clotting time after recalcification is shortened to 26 to 33 seconds by the addition of negatively charged phospholipids and a particulate substance such as kaolin (aluminum silicate); this is termed the activated partial thromboplastin time (aPTT). Alternatively, recalcified plasma will clot in 12 to 14 seconds after addition of "thromboplastin" (a mixture of tissue factor and phospholipids); this is termed the prothrombin time (PT).

Two pathways of coagulation are recognized. An individual with a prolonged aPTT and a normal PT is considered to have a defect in the intrinsic coagulation pathway, because all of the components of the aPTT test (except kaolin) are intrinsic to the plasma. A patient with a prolonged PT and a normal aPTT has a defect in the extrinsic coagulation pathway, since thromboplastin is extrinsic to the plasma. Prolongation of both the aPTT and the PT suggests a defect in a common pathway.

Natural Anticoagulant Mechanisms. Platelet activation and coagulation normally do not occur within an intact blood vessel (Edelberg et al., 2001). Thrombosis is prevented by several regulatory mechanisms that require a normal vascular endothelium. Prostacyclin (prostaglandin I2; PGI2), a metabolite of arachidonic acid, is synthesized by endothelial cells and inhibits platelet aggregation and secretion. Antithrombin is a plasma protein that inhibits coagulation factors of the intrinsic and common pathways (see below). Heparan sulfate proteoglycans synthesized by endothelial cells stimulate the activity of antithrombin. Protein C is a plasma zymogen that is homologous to II, VII, IX, and X; its activity depends on the binding of Ca2+ to Gla residues within its amino-terminal domain. Activated protein C, in combination with its nonenzymatic Gla-containing cofactor (protein S), degrades cofactors Va and VIIIa and thereby greatly diminishes the rates of activation of prothrombin and factor X (Esmon, 2003). Protein C is activated by thrombin only in the presence of thrombomodulin, an integral membrane protein of endothelial cells. Like antithrombin, protein C appears to exert an anticoagulant effect in the vicinity of intact endothelial cells. Tissue factor pathway inhibitor (TFPI) is found in the lipoprotein fraction of plasma. When bound to factor Xa, TFPI inhibits factor Xa and the factor VIIa-tissue factor complex. By this mechanism, factor Xa may regulate its own production
Philip W. Majerus and Douglas M. Tollefsen
key words: blood, Prostacyclin, Anticoagulant Mechanisms, Fibrinolysis and Thrombolysis